Methamphetamine derivatives and conjugates for immunoassay

ABSTRACT

Compounds of methamphetamine derivatives having a meta-substituted alkyl linker on the benzene ring and a protective group on the nitrogen of the methamphetamine hapten. Such compounds have the structure 
                         
wherein R 1  is an alkyl linker comprising 2-15 carbon atoms and 0-6 heteroatoms, R 2  is a leaving group, and R 3  is a protecting group.

FIELD OF THE INVENTION

The present invention relates generally to the field of immunoassaymethods for determination of drugs of abuse in biological samples, inparticular, to immunoassay methods for determination ofamphetamine-class drugs, and more particularly, to methamphetaminederivatives and conjugates useful therefor.

BACKGROUND OF THE INVENTION

The use and abuse of a class of illicit designer drugs known commonly as“ecstasy-class drugs” have increased significantly in recent years.These compounds, which are derivatives of amphetamine distinguished byhaving a fused methylenedioxy phenyl ring system, include MDA(3,4-methylenedioxyamphetamine), MDMA, also known as “Ecstasy”(3,4-methylenedioxy-N-methylamphetamine), MDEA, also known as “Eve”(3,4-methylenedioxy-N-ethylamphetamine), BDB(3,4-methylenedioxyphenyl-2-butanamine), and MBDB(3,4-methylenedioxyphenyl-N-methylbutanamine).

Heretofore, methods for the detection of ecstasy-class drugs haveprimarily involved immunoassays originally developed for the detectionof amphetamine and/or methamphetamine. The detection of an ecstasy-classdrug by such assays relies on the limited cross-reactivities that maycoincidentally exist between the ecstasy-class drug and the amphetamineand/or methamphetamine antibodies. A positive result obtained by such anassay may still not indicate which particular substance or member of themethylenedioxy (MD) class of derivatives is present in a sample.

In general, amphetamine and methamphetamine immunoassays are relativelyinsensitive to, and non-specific for, ecstasy-class drugs. Such assaysshow particularly limited recognition for the MDEA derivative.

The present invention is directed to remedying these and other problemsrelating to the use of conventional amphetamine and/or methamphetamineimmunoassays for the detection of members of the methylenedioxy class,or ecstasy-class, drugs.

In testing for drugs of abuse, immunoassays, particularly competitivebinding immunoassays, have proven to be especially advantageous. Incompetitive binding immunoassays, an analyte in a biological samplecompetes with a labeled reagent, or analyte analog, or tracer, for alimited number of receptor binding sites on antibodies specific for theanalyte and analyte analog. Enzymes such as β-galactosidase andperoxidase, fluorescent molecules such as fluorescein compounds,radioactive compounds such as ¹²⁵I, and microparticles are commonlabeling substances used as tracers. The concentration of analyte in thesample determines the amount of analyte analog which will bind to theantibody. The amount of analyte analog that will bind is inverselyproportional to the concentration of analyte in the sample because theanalyte and the analyte analog each bind to the antibody in proportionto their respective concentrations. The amount of free or bound analyteanalog can then be determined by methods appropriate to the particularlabel being used.

Currently popular are automated assays based on kinetic interaction ofmicroparticles in a solution as measured by changes in lighttransmission. In the absence of sample drug, free antibody binds todrug-microparticle conjugates causing the formation of particleaggregates. As the aggregation reaction proceeds in the absence ofsample drug, absorbance increases. When a sample contains a drug inquestion, the drug competes with a particle-bound drug derivative forfree antibody. Antibody bound to sample drug is no longer available topromote particle aggregation, and subsequent particle lattice formationis inhibited. The presence of sample drug diminishes increasingabsorbance in proportion to concentration of drug in the sample. Sampledrug content is determined relative to the value obtained for a knowncutoff concentration of drug.

A problem in assays for amphetamine-class drugs is that prior artmethods do not provide sufficient cross-reactivity with and displacementfrom antibodies to measure all the amphetamine-class analytes in asample, especially amphetamine, methamphetamine,3,4-methylenedioxyamphetamine (MDA),3,4-methylenedioxy-N-methylamphetamine (MDMA), and3,4-methylenedioxy-N-ethylamphetamine (MDEA), with sufficientsensitivity. The methamphetamine derivatives of the present inventionovercome such problems by providing the required cross-reactivity withand displacement from methamphetamine antibodies to sensitivemeasurement of amphetamine, methamphetamine, and ecstasy-classcompounds.

Another problem overcome by the present invention is that of unwantedconjugation of haptens through the free amino group on themethamphetamine hapten. The derivatives of the present invention avoidthis problem.

In U.S. Pat. No. 5,135,863 issued Aug. 4, 1992, Hu et al. describeconjugates comprising a label bound to an analog of amphetamine ormethamphetamine via a thiol linkage. The conjugates further comprise alinker bonded to the benzene ring at the meta- or para-position, and theamphetamine nitrogen is unprotected.

In U.S. patent application Ser. No. 2001/0051158 published Dec. 13,2001, Owens et al. describe immunochemical haptens which aremeta-position derivatives of methamphetamine. The free amino group onthe methamphetamine hapten is unprotected.

SUMMARY OF THE INVENTION

Compounds of the present invention are methamphetamine derivativeshaving a meta-substituted alkyl linker on the benzene ring and aprotective group on the nitrogen of the methamphetamine hapten. Suchcompounds have the structure

wherein R₁ is an alkyl linker comprising 2-15 carbon atoms and 0-6heteroatoms, R₂ is a leaving group, and R₃ is a protecting group.

A preferred compound of the invention is4-{[4-(3-{(S)-2-[methyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-phenoxy)-butyrylamino]-methyl}-benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester having the structure

This compound, when conjugated to aminodextran followed by deprotectionof the N-trifluoroacetate group, provides a conjugate that, when used inan immunoassay wherein it competes with free methamphetamine for bindingto a methamphetamine-specific antibody immobilized on a microparticle,provides a standard curve having a good fit with the correspondingamphetamine-aminodextran-antibody system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation showing synthesis of (2) asdescribed in Example 2.

FIGS. 2 and 3 are schematic representations of the synthesis of acompound of the present invention,4-(3-{(S)-2-[(2,2-dimethyl-propionyl)-methyl-amino]-propyl}-phenoxy)-butyricacid 2,5-dioxo-pyrrolidin-1-yl ester (8).

FIG. 4 is a schematic representation showing synthesis of4-{[4-(3-{(S)-2-[methyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-phenoxy)-butyrylamino]-methyl}-benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester (10) as described in Example 10.

FIG. 5 is a schematic representation showing conjugation of a compoundof the present invention with BSA and deprotection of the nitrogen togive (11) as described in Example 11.

FIG. 6 is a schematic representation showing conjugation of a compoundof the present invention with aminodextran followed by deprotection ofthe N-trifluoroacetate group.

FIG. 7 is a schematic representation showing synthesis of (15) asdescribed in Example 12.

FIG. 8 is a schematic representation showing synthesis of (17) asdescribed in Example 13.

FIG. 9 is a schematic representation showing synthesis of (18) asdescribed in Example 14.

FIG. 10 is a schematic representation showing synthesis of (19) asdescribed in Example 15.

FIG. 11 is a dose response curve generated using the aminodextranconjugate (12) of the present invention with methamphetamineantibody-coated particles.

FIG. 12 is a dose response curve generated using the aminodextranconjugate (12) of the present invention with N-ethylamphetamineantibody-coated particles.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this description and in the appended claims, the followingdefinitions are to be understood.

The term “hapten” refers to a partial or incomplete antigen. Haptens areprotein-free substances, mostly low molecular weight substances, whichare not capable of stimulating antibody formation but which do reactwith antibodies. Amphetamine, methamphetamine,methylenedioxymethamphetamine, and other amphetamine-class compounds arehaptens.

The term “activated hapten” refers to a hapten that has been providedwith an available reaction site, for example, by the attachment of alinking group carrying a reactive moiety, that can be used to connectthe hapten to a carrier, immunogen, label, tracer or other moiety.

The term “analyte” refers to any substance or group of substances, thepresence or amount of which is to be determined. As used herein, theterm analyte subsumes the term “antigen,” which refers to any compoundthat can bind to an antibody.

The terms “amphetamine” and “amphetamine-class drugs” as used hereinrefer to amphetamine, methamphetamine, amphetamine analogues ofmethylenedioxyphenylalkylamines, also known as “designer” amphetamines,e.g., methylenedioxymethamphetamine (MDMA, also known as Ecstasy), andmetabolites of these drugs.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “conjugate” refers to any substance formed from the joiningtogether of two parts. Representative conjugates in accordance with thepresent invention include those formed by the joining together of asmall molecule and a large molecule, such as a protein. The termconjugate subsumes the term immunogen.

As used herein, a “linking group” or “linker” refers to a portion of achemical structure which connects two or more substructures such ashaptens, carriers, immunogens, labels, tracers or other linkers. Alinking group has at least 1 uninterrupted chain of atoms other thanhydrogen (or other monovalent atoms) extending between thesubstructures. The atoms of a linking group and the atoms of a chainwithin a linking group are themselves connected by chemical bonds.Linkers may be straight or branched, saturated or unsaturated, carbonchains. They may also include one or more heteroatoms within the chainor at termini of the chains. By “heteroatoms” is meant atoms other thancarbon which are chosen from the group consisting of oxygen, nitrogenand sulfur. Linking groups may also include cyclic or aromatic groups aspart of the chain or as a substitution on one of the atoms in the chain.

The number of atoms in a linking group or linker is determined bycounting the atoms other than hydrogen. The number of atoms in a chainwithin a linking group is determined by counting the number of atomsother than hydrogen along the shortest route between the substructuresbeing connected. Linking groups may be used to activate, e.g., providean available site on a hapten for synthesizing a conjugate of a haptenwith a label or carrier.

The term “alkyl group” refers to any straight, branched, cyclic,acyclic, saturated or unsaturated carbon chain. Representative alkylgroups include alkanes, alkenes, alkynes, cycloalkanes, cycloalkenes,cycloalkynes, aryls, and the like, and combinations thereof.

The phrase “optionally substituted” refers to the optional attachment ofone or more substituents onto an alkyl group.

The term “leaving group” refers to any chemical moiety of a substratethat can be displaced by a reagent reacted therewith. Suitable leavinggroups include, but are not limited to, halides, mesylates, tosylates,alkoxys, quaternary ammonium salts, and the like. Preferred leavinggroups for use in accordance with the presently preferred embodimentsare provided by activated esters, e.g., trifluoroethoxy esters,N-hydroxysuccinimide esters, p-nitrophenyl esters, pentafluorophenylesters, imidazolyl esters, and N-hydroxybenzotriazolyl esters, wherebythe oxygen-containing portion of the ester that is attached to thecarbonyl carbon is displaced in the course of the reaction.

The term “protecting group” refers to any moiety that is attached to areactive atom or center in order to alter its usual reactivity. Suitableprotecting groups include but are not limited to those described in thetreatise entitled Protective Groups in Organic Synthesis, 3^(rd) Editionby Theodora W. Greene and Peter G. M. Wuts (John Wiley & Sons, Inc., NewYork, 1999). Various protecting groups for the nitrogen of amines areknown in the art, among which trifluoroacetyl is a presently preferrednitrogen protecting group. Examples of other preferred protecting groupsinclude t-Boc (tert-butyloxycarbonyl) and CBZ (benzyloxycarbonyl).

The term “analyte analog” refers to any substance or group ofsubstances, such as may be employed in a competitive immunoassay, whichbehaves similarly to an analyte with respect to binding affinity to anantibody. Representative analyte analogs include drugs and isomersthereof, drug derivatives, hormones, polypeptides, nucleotides, and thelike.

The phrase “detecting an analyte” refers to any quantitative,semi-quantitative, or qualitative method, as well as to all othermethods for determining an analyte in general, and an amphetamine drugin particular. For example, a method that merely detects the presence orabsence of an amphetamine drug in a sample lies within the scope of thepresent invention, as do methods that provide data as to the amount orconcentration of the drug in the sample. The terms detecting,determining, identifying, and the like are used synonymously herein, andall are within the scope of the present invention.

A “label”, “detector molecule”, or “tracer” is any molecule whichproduces, or can be induced to produce, a detectable signal. The labelcan be conjugated to an analyte, immunogen, antibody, or to anothermolecule such as a receptor or a molecule that can bind to a receptorsuch as a ligand, particularly a hapten. Non-limiting examples of labelsinclude radioactive isotopes, enzymes, enzyme fragments, enzymesubstrates, enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes,chemiluminescers, luminescers, sensitizers, non-magnetic or magneticparticles, solid supports, liposomes, ligands, and receptors.

The term “reagent kit,” or “test kit,” refers to an assembly ofmaterials that are used in performing an assay. The reagents can beprovided in packaged combination in the same or in separate containers,depending on their cross-reactivities and stabilities, and in liquid orin lyophilized form. The amounts and proportions of reagents provided inthe kit can be selected so as to provide optimum results for aparticular application. A reagent kit embodying features of the presentinvention comprises antibodies specific for amphetamine compounds. Thekit may further comprise ligands of the analyte and calibration andcontrol materials. The reagents may remain in liquid form or may belyophilized.

The phrase “calibration and control materials” refers to any standard orreference material containing a known amount of an analyte to bemeasured. A sample suspected of containing an analyte and thecorresponding calibration material are assayed under similar conditions.The concentration of analyte is calculated by comparing the resultsobtained for the unknown specimen with the results obtained for thestandard. This is commonly done by constructing a calibration or doseresponse curve.

Compounds of the present invention are methamphetamine derivativeshaving a meta-substituted alkyl or alkoxy linker on the benzene ring anda protective group at the nitrogen. Such compounds have the structure

wherein R₁ is an alkyl linker comprising 2-15 carbon atoms and 0-6heteroatoms, R₂ is a leaving group, and R₃ is a protecting group.

A preferred compound of the invention has the structure

Such a compound, when conjugated to carrier molecules such asaminodextran followed by deprotection of the N-trifluoroacetate group,provides a conjugate that, when used in an immunoassay method whereinthe conjugate competes with free methamphetamine for binding to amethamphetamine-specific antibody immobilized on a microparticle,provides a standard curve having good correlation with the correspondingamphetamine-aminodextran-antibody system. The combination also providessuitable cross reactivities with both desired (high cross reactivity)and undesired (low cross reactivity) analytes. When conjugated to bovineserum albumin at a low substitution ratio, such a compound alsofurnishes a conjugate suitable for the screening of methamphetamineantibodies.

The derivatives carry a protective group at the nitrogen. Such aprotective group allows for the easy reaction of the active ester moietyof the derivatives with, typically, amines of carrier compounds to formstable amide bonds without the amine of the derivative itselfinterfering. The protective group is then subsequently readily removedto provide the desired unprotected amine moiety corresponding to theamine of the free drug.

The position of substitution and ether linkage of the linking group werealso found to be advantageous in moderating the affinity of the antibodyfor the derivative conjugate, while the use of the aminomethylbenzoatemoiety also aids in both easier characterization of the derivative(greater UV absorbance) as well as enhancing the solid nature of thederivatives (as opposed to gums or liquids).

It was quite surprisingly found that the derivative of the presentinvention is recognized by two antibodies raised in response toimmunogens which are structurally quite distinct from the novelderivative. One difference is the position of substitution, which ispara in the two immunogens but meta in the conjugate. Another differenceis that the linker used for the immunogens does not have an oxygen atomcoming off the benzene ring as does the conjugate. Finally, the nitrogenin the immunogens is bonded to a methyl group whereas the nitrogen inthe conjugate of the invention is bonded to an ethyl group.

SPECIFIC EMBODIMENTS

In the examples that follow, boldface numbers refer to the correspondingstructure in the drawings.

Flash chromatography was carried out on silica gel 60 (230-400 mesh, EMScience). Thin layer chromatography was performed on silica gel plates(0.25 mm, EM Science, Cat #5717-5) and visualized under an ultravioletlamp.

Solvents were obtained from J.T. Baker Company unless otherwise stated.Ethyl acetate (EtOAc), hexanes (Hex) methanol (MeOH), and methylenechloride (CH₂Cl₂) were used as received for chromatography and reactionwork-ups. Dry CH₂Cl₂ was obtained by boiling over calcium hydride underargon and under reflux. Dry tetrahydrofuran (THF) was obtained byboiling over sodium-benzophenone under argon and under reflux. Drydimethylformamide (DMF) and dry dimethylsulfoxide (DMSO) were obtainedin Sure/Seal™ bottles from Aldrich Chemical Company.

Reagents and chemicals were obtained from either Sigma-Aldrich ChemicalCompany or from Fluka Chemicals unless otherwise stated.

Proton nuclear magnetic resonance spectra (¹H-NMR) were obtained on aVarian Gemini 2000 (200 MHz) equipped with a Sun/Sparc work station.Optical rotations were performed on a Perkin Elmer 341 polarimeter.

3N perchloric acid was prepared by diluting 70% perchloric acid (11.7N)

Liquid chromatography mass spectra (LC-MS) and high performance liquidchromatography spectra (HPLC) were obtained on an Agilent HP1100 LC/MSsystem equipped with a diode array detector and quaternary pump. ForLC-MS spectra/analyses, the chromatographic stream was portedpost-column into the MSD detector. Unless otherwise stated, theanalytical column used was a Vydac 218TP54 analytical column (300 Å, 5μ) equipped with a Phenomenex guard module (KJO-4282). Unless otherwisestated, runs were performed using 0.1% TFA-MeCN (C) in 0.1% TFA-H₂O (A)with a solvent gradient of 5% (0 min) to 100% (20 min) to 5% (25 min) of(C) in (A).

EXAMPLE 1 Synthesis of compound (1),[(S)-2-(3-methoxy-phenyl)-1-methyl-ethyl]-((S)-1-phenyl-ethyl)-amine

A solution of 6.0 g (0.0365 mol) of 3-methoxyphenylacetone (Trans WorldChem.) and 4.47 g (0.0369 mol) of S-(−)-α-phenylethylamine (Fluka) in100 ml of benzene (Acros) was treated with 6.0 g of 4 Å molecular sievesand heated to reflux for 3 hrs. The mixture was filtered andconcentrated at reduced pressure. The residue was dissolved in 100 ml ofabsolute ethanol (Aldrich), placed in a 500 ml Parr bottle containing2.4 g of Raney nickel (50% slurry in H₂O) and hydrogenated at 50 PSI for24 hrs. The catalyst was filtered off through CELITE and the filtratewas concentrated at reduced pressure. The residue was purified by flashchromatography on 800 g of silica gel using 3% methanol-CH₂Cl₂ as eluentto yield 5.0 g of product as a pale yellow oil. ¹H-NMR (CDCl₃)compatible; LC-MS: t_(R) 11.4 min, observed M+H 270.2; [α]_(D) −36.6°(589 nm, c=l, CHCl₃).

EXAMPLE 2 Synthesis of compound (2),(S)-2-(3-methoxy-phenyl)-1-methyl-ethylamine

A 500 ml Parr bottle was charged with 550 mg of 10% Pd/C and a solutionof 5.0 g (0.0186 mol) of compound (1) in 100 ml of methanol andhydrogenated at 50 PSI for 48 hrs. The catalyst was filtered off throughCELITE and the filtrate was concentrated at reduced pressure to a yellowoil. This was purified by flash chromatography on a short column ofsilica gel (4 cm W, 5 cm H) using 10% methanol-CH₂Cl₂ to remove residualcatalyst to yield 2.84 g of yellow oil. This was dissolved in CH₂Cl₂ andfiltered to remove residual silica gel, then concentrated at reducedpressure and pumped at high vacuum overnight to yield 2.5 g of yellowoil. ¹H-NMR (CDCl₃) compatible; LC-MS: t_(R) 8.05 min (Chiralcel OD-RH,Daicel Chemical Industries; gradient of 5% (at 0 min) to 85% (at 17.5min) of 0.1% TFA/acetonitrile in 0.1% TFA/water; 0.5 mL/min flow rate),observed M+H 166.1, one peak; HPLC: t_(R) 30.0 min (d-isomer), 33.7 min(l-isomer), ratio d/l=97.2/2.8 at 200 nm (Crownpak CR(+), DaicelChemical Industries; isocratic 1 ml/min of aq perchloric acid pH 1.35(˜0.2%) containing 5% MeOH); [α]_(D) +31.7° (589 nm, c=1, CHCl₃).

EXAMPLE 3 Synthesis of compound (3),2,2,2-trifluoro-N-[(S)-2-(3-methoxy-phenyl)-1-methyl-ethyl]-acetamide

A solution of 2.4 g (0.0145 mol) of compound (2) in 24 ml of pyridine(Aldrich) under argon was cooled to −20° C. and treated with a solutionof 3.2 ml of trifluoroacetic anhydride in 36 ml of ether (E. M. Science)and stirred at −20° C. for 15 min. The mixture was extracted with 3×100ml of ethyl acetate. The combined ethyl acetate extracts were washedwith 2×150 ml of saturated brine solution, dried over anhydrous Na₂SO₄and concentrated at reduced pressure to yield 3.79 g of yellow solid.¹H-NMR (CDCl₃) compatible.

EXAMPLE 4 Synthesis of compound (4),2,2,2-trifluoro-N-[(S)-2-(3-hydroxy-phenyl)-1-methyl-ethyl]-acetamide

A pressure bomb containing 3.77 g (0.0144 mol) of compound (3) and 70.0g (0.6057 mol) of pyridine-HCl was purged with argon and heated at 210°C. for 45 min. The solids melted and became homogeneous. The reactionmixture was allowed to cool, at which time it solidified. The solid wasdissolved in H₂O and poured into 400 ml of ice water. This was extractedwith 5×250 ml of CH₂Cl₂. The CH₂Cl₂ extracts were combined, dried overanhydrous Na₂SO₄ and concentrated at reduced pressure, then pumped athigh vacuum overnight to yield 3.34 g of yellow oil. ¹H-NMR (CDCl₃)compatible; LC-MS compatible.

In another run, similar reaction of 320 mg of compound (3) and 5.94 g ofpyridine hydrochloride in a pressure bomb gave, after similar work-up,213 mg of compound (4) which had: ¹H-NMR compatible; LC-MS: t_(R) 11.4min, observed M+H 248.1, M+Na 270.1.

EXAMPLE 5 Synthesis of compound (5),4-{3-[(S)-2-(2,2,2-trifluoro-acetylamino)-propyl]-phenoxy}-butyric acidethyl ester

A mixture of 1.08 g (0.027 mol) of NaH (60% dispersion in mineral oil)and 10 ml of anhydrous DMF (Aldrich) under argon was treated with asolution of 3.3 g (0.0133 mol) of compound (4) in 33 ml of anhydrous DMF(Aldrich) added dropwise over a 5 min. time period. The mixture wasstirred at ambient temperature for 10 min. then treated with 3.4 ml(0.0238 mol) of ethyl-4-bromobutyrate (Fluka) and stirred at roomtemperature overnight. The mixture was poured into 250 ml of 1Mpotassium phosphate buffer pH 7, then extracted with 2×250 ml of ethylacetate. The organic extracts were combined and washed with 2×150 ml of1M potassium phosphate buffer pH 7, dried over anhydrous Na₂SO₄ andconc. at reduced pressure. The residue was purified by flashchromatography on 250 g of silica gel using 30% EtOAc-hexane as eluentto yield a pale yellow oil which crystallized on standing to yield 3.9 gof a white solid. ¹H-NMR (CDCl₃) compatible; LC-MS: t_(R) 15.7 min,observed M+H 362.1, M+H₂O 379.1, M+Na 384.1.

EXAMPLE 6 Synthesis of compound (6),4-(3-{(S)-2-[methyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-phenoxy)-butyricacid ethyl ester

A solution of 3.8 g (0.0105 mol) of compound (5) in 72 ml of anhydrousDMF (Aldrich) under argon was treated with 9.5 ml (0.1526 mol) ofiodomethane and 11.74 g (0.0507 mol) of silver (I) oxide and stirred at40° C. overnight. The mixture was filtered through CELITE, the filtercake was washed with 450 ml of ethyl acetate, and the filtrate wasfiltered again to remove a white precipitate. The filtrate was washedwith 2×250 ml of H₂O. The H₂O washes were combined and extracted with200 ml of ethyl acetate. The organic extracts were combined, dried overanhydrous Na₂SO₄, concentrated at reduced pressure and pumped at highvacuum overnight to yield 3.84 g of pale yellow oil. ¹H-NMR (CDCl₃)compatible, ˜2:1 ratio of rotamers.

EXAMPLE 7 Synthesis of compound (7),4-(3-{(S)-2-[methyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-phenoxy)-butricacid

A solution of 3.8 g (00101 mol) of compound (6) in 50 ml of distilledTHF was treated with 50 ml of 3N perchloric acid (prepared from 70%perchloric acid obtained from Aldrich) and heated at 50° C. for 4 hrs.The mixture was cooled and extracted with 2×200 ml of ethyl acetate. Theorganic extracts were combined, washed with 4×200 ml of H₂O, 2×200 ml ofsaturated brine solution, dried over anhydrous Na₂SO₄, concentrated atreduced pressure and pumped at high vacuum overnight to yield 3.5 g ofpale yellow oil. ¹H-NMR (CDCl₃) compatible, ˜2:1 ratio of rotamers.

EXAMPLE 8 Synthesis of compound (8),4-(3-{(S)-2-[(2,2-dimethyl-propionyl)-methyl-amino]-propyl}-phenoxy)-butyricacid 2,5-dioxo-pyrrolidin-1-yl ester

A solution of 3.5 g (0.0101 mol) of compound (7) in 220 ml of anhydrousCH₂Cl₂ under argon was treated with 1.4 g (0.0122 mol) of NHS and 2.4 g(0.0125 mol) of EDC (Sigma) and stirred at room temperature overnight.The mixture was washed with 200 ml of 0.1N HCl, 2×200 ml of H₂O, 2×200ml of saturated NaHCO₃ solution, 200 ml of saturated brine solution,dried over anhydrous Na₂SO₄ and concentrated at reduced pressure toyield 4.42 g of clear oil. ¹H-NMR (CDCl₃) compatible, ˜3:2 ratio ofrotamers.

EXAMPLE 9 Synthesis of compound (9),4-{[4-(3-{(S)-2-[methyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-phenoxy)-butyrylamino]-methyl}-benzoicacid

A mixture of 1.6 g (0.0106 mol) of 4-aminomethylbenzoic acid in 60 ml ofH₂O and 120 ml distilled THF was treated with 10 ml of 1N NaOH. Theresulting solution was then treated with a solution of 4.4 g (0.01 mol)of compound (8) in 120 ml of distilled THF. The reaction was thentreated with 4 ml of 1N NaOH added 1 ml at a time to maintain pH 9. Thereaction was stirred at room temperature for 1 hr. The THF was removedat reduced pressure and the aqueous residue was neutralized to pH 6 with2N HCl. This was extracted with 2×250 ml of ethyl acetate. The organicextracts were combined, dried over anhydrous Na₂SO₄ and concentrated atreduced pressure to yield 4.65 g of white solid. ¹H-NMR (CDCl₃)compatible, ˜3:2 ratio of rotamers.

EXAMPLE 10 Synthesis of compound (10),4-{[4-(3-{(S)-2-[methyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-phenoxy)-butyrylamino]-methyl}-benzoicacid 2,5-dioxo-pyrrolidin-1-yl ester

A solution of 1.0 g (0.002 mol) of compound (9) in 100 ml of anhydrousCH₂Cl₂ under argon was treated with 300 mg (0.0026 mol) of NHS and 480mg (0.0025 mol) of EDC (Sigma) and stirred at room temperatureovernight. The mixture was washed with 100 ml of 0.1N HCl, 2×100 ml ofH₂O, 2×100 ml of saturated NaHCO₃ solution, 100 ml of saturated brinesolution, dried over anhydrous Na₂SO₄ and concentrated at reducedpressure to yield 1.13 g of white amorphous solid. ¹H-NMR (CDCl₃)compatible, ˜3:2 ratio of rotamers; LC-MS: t_(R) 14.5 min, observed M+H578.2, M+Na 600.2; [α]_(D) +15.7° (589 nm, c=1, CHCl₃).

EXAMPLE 11 Synthesis of compound (11), deprotected4-(3-{(S)-2-[(2,2-dimethyl-propionyl)-methyl-amino]-propyl}-phenoxy)-butyricacid 2,5-dioxo-pyrrolidin-1-yl ester conjugate with BSA

A solution of 1.0 g of bovine serum albumin (BSA; Pentex Fraction V;Miles Inc., Kankakee, Ill., USA) in 16 ml of 50 mM potassium phosphate(KPi) pH 7.5 was cooled in an ice bath and treated with 22 ml of DMSOadded dropwise slowly. After addition was complete, a solution of 13.4mg of compound (8) in 2 ml of DMSO was added dropwise and the reactionallowed to attain RT with stirring. A reference sample of 100 mg of BSAin 1.6 ml of 50 mM KPi pH 7.5 and 2.4 ml of DMSO was also prepared in asimilar manner but no hapten was added. After stirring overnight boththe reaction and reference sample were separately transferred todialysis tubing (SpectraPor 7; 10,000 molecular weight cutoff) anddialyzed against 60% DMSO −50 mM KPi, pH 7.5 (twice at RT/4 h each; onceat RT overnight) then sequentially against 40% to 20% to 10% DMSO −50 mMKPi, pH7.5, then against 50 mM KPi pH7.5. Dialysis was continued against50 mM potassium carbonate (four changes) at RT for 4 d to deprotect thetrifluoroacetyl group, then against 50 mM KPi pH7.5 at ˜4° C. overseveral days (six changes). The retentates were recovered to give theconjugate (11) as a grayish solution (68 ml), as well as the referenceBSA. Coomassie Blue protein assay, using the reference BSA [proteinconcentration by UV] to generate the standard curve, indicated 14.0mg/ml protein for conjugate (11).

EXAMPLE 12 Synthesis of compound (15),[2-(3-{(S)-2-[methyl-(2,2,2-trifluoro-acetyl)-amino]-propyl}-phenoxy)-ethyl]-carbamicacid tert-butyl ester

Compound (13),2,2,2-trifluoro-N-[(S)-2-(3-methoxy-phenyl)-1-methyl-ethyl]-N-methyl-acetamide.A solution of 384 mg of compound (3) in 10 ml of dry dimethylformamide(DMF) under argon was treated with 1.33 ml of iodomethane and 1.64 g ofsilver (I) oxide and stirred at 40° C. overnight. The mixture wasfiltered through CELITE and the filter cake washed with ethyl acetate.The filtrate was filtered again to remove a white precipitate. Thefiltrate was washed with 2×50 ml of H₂O. The aqueous washes werecombined and extracted with 50 ml of ethyl acetate. The ethyl acetateportions were combined, dried over anhydrous sodium sulfate, andconcentrated at reduced pressure to yield 330 mg of yellow oil. This waschromatographed on 50 g of silica gel using CH₂Cl₂ as eluent to yield220 mg of the product, compound (13), as a pale yellow oil. LC-MS: t_(R)15.4 min, observed M+H 276.0, M+Na 298.0.

Compound (14),2,2,2-trifluoro-N-[(S)-2-(3-hydroxy-phenyl)-1-methyl-ethyl]-N-methyl-acetamide.A mixture of 220 mg of compound (13) and 3.87 g of pyridinehydrochloride were heated at 210° C. in a pressure bomb for 1 hr. Theresulting melt was allowed to cool and was dissolved in water. This wasextracted with 5×50 ml of CH₂Cl₂. The combined CH₂Cl₂ extracts weredried over anhydrous sodium sulfate and concentrated at reduced pressureto yield 100 mg of compound (14) as a yellow oil. LC-MS: t_(R) 12.9 min,observed M+H 262.0, M+Na 284.0.

Compound (15). A solution of 25 mg of compound (14) in 10 ml of dryacetone was treated with a solution of 2-(BOC-amino)ethyl bromide in 1ml of dry DMF, 77 mg of anhydrous potassium carbonate, and a catalyticamount of 18-crown-6. The mixture was heated at 60° C. overnight. Themixture was cooled and diluted with CH₂Cl₂, washed 3 times with H₂O,dried over anhydrous sodium sulfate, and concentrated at reducedpressure to a red oil containing compound (15). LC-MS: tR 16.5 min,observed M+Na 427.1.

EXAMPLE 13 Synthesis of Compound (17)

Compound (15) is treated with trifluoroacetic acid and stirred at roomtemperature for 2 hrs. The mixture is concentrated at reduced pressureto give the amine compound (16).

A solution of biphenyl-di-carboxylic acid NHS ester, compound (20)(Ghoshal et al., U.S. Pat. No. 6,794,496), in dry THF under argon istreated with a solution of 1 equivalent of compound (16) in dry THF with2 equivalents of triethylamine added dropwise. The mixture is stirred atroom temperature overnight. The mixture is concentrated at reducedpressure. Purification by chromatography gives compound (17).

EXAMPLE 14 Synthesis of Compound (18)

A solution of terephthalic acid di-(N-hydroxysuccinimide) ester compound(21) (Ghoshal et al., European Patent Application 1,148,339) in dry THF,under argon, is treated with a solution of 1 equivalent of compound (16)in dry THF with 2 equivalents of triethylamine added dropwise. Themixture is stirred at room temperature overnight. The mixture isconcentrated at reduced pressure. Purification by chromatography givescompound (18).

EXAMPLE 15 Synthesis of Compound (19)

A solution of 4-isothiocyanatobenzoyl chloride, compound (22) (Ghoshalet al., U.S. Pat. No. 6,794,496), in dry THF under argon is cooled withan ice bath and is treated with a solution of 1 equivalent of compound(16) in dry THF with 2 equivalents of triethylamine added dropwise. Themixture is stirred at room temperature overnight. The mixture isconcentrated at reduced pressure. Purification by chromatography givescompound (19).

EXAMPLE 16 Assay Using Conjugate of Present Invention withMethamphetamine Antibody

The methamphetamine-aminodextran conjugate, compound (12), was preparedas follows: Ninety mg of aminodextran were dissolved in 5 ml DMSO. Tenmg of compound (10) were dissolved in 1 ml DMSO and added dropwise tothe solution of the polymer carrier and stirred at room temperatureovernight. The mixture was placed into 10,000 MW cut-off dialysis tubing(Pierce Snakeskin™) and dialyzed in 1 liter of 80% DMSO overnight atroom temperature. This was then dialyzed using a step-down gradient in 1liter of 60% DMSO, 1 liter of 40% DMSO, 1 liter of 20% DMSO, 4 liters ofdeionized water, for at least 3 each at room temperature. This was thendialyzed with 1 liter of 100 mM K₂CO₃, adjusted to pH 13 with KOH, for 2days with 4 changes of buffer. A final dialysis step included 4 litersof deionized water for at least 2 days with 4 changes. The conjugate wasthen lyophilized.

A conjugate reagent, pH 7.1, was then prepared containing 0.125 μg/mlmethamphetamine conjugate (12), 144 mMpiperazine-1,4-bis(2-ethanesulfonic acid) disodium salt, 31 mMpiperazine-1,4-bis(2-ethanesulfonic acid), 0.1% (w/v) BSA, 0.09% (w/v)sodium azide, and 1.5% (w/v) polyacrylic acid.

An antibody-microparticle reagent was prepared as follows: 100 ml of 50μg/ml monoclonal antibody specific for methamphetamine was incubatedwith 100 ml 1% 0.201 μm latex particles in 50 mM MES buffer, pH 6.5,overnight at room temperature. The particles were blocked with 10 ml of100 mg/ml BSA in 50 mM MES buffer, pH 6.5, for 2 hours. Latex was washedwith 50 mM MOPS buffer, pH 7.1, using tangential flow filtration system.This antibody was raised in response to the immunogen described in U.S.Pat. No. 5,501,987 (column 3, structure 2 where the protein is bovinethyroglobulin).

A microparticle reagent, pH 7.1, was then prepared containing 0.1%solids antibody-coated microparticles, 25 mM 3-morpholinopropanesulfonicacid, 25 mM 3-morpholinopropanesulfonic acid, sodium salt, 0.1% (w/v)BSA, and 0.09% (w/v) sodium azide.

The assay was carried out on a Roche/Hitachi 917 analyzer. Ten μl ofsample was pipetted into a cuvette and was immediately followed by theaddition of 180 μl conjugate reagent (R1). The mixture was incubated for˜90 seconds, and 80 μl of microparticle reagent (R2) were added to thecuvette. The reaction mixture was incubated at 37° C. for approximately8 min, during which time the particle agglutination reaction wasmonitored at 505 nm. A standard curve was devised based on a 2-point endmeasurement after R2 addition.

Percent cross-reactivity was determined in an immunoassay. A standardcurve can was established using known amounts of the target analytemethamphetamine. Known amounts of various amphetamine-related drugs werebe analyzed as samples in the immunoassay, and apparent concentrationsof these drugs were generated from the standard curve. Apparentconcentration of the drug, divided by actual concentration andmultiplied by 100, is the percent cross-reactivity of this drug. Thestandard (dose response) curve thus generated is shown in FIG. 11.

The cross-reactivity of the methamphetamine-coated particle withconjugate (12) is given in the table below.

Compound Conc (ng/ml) tested Result % Cross-reactivity d-pseudoephedrine100,000 326 0.3 l-ephedrine 100,000 197 0.2 Tyramine 100,000 169 0.2Phentermine 100,000 113 0.1 BDB 2,000 69 3 MBDB 1,000 467 47 MDA 600 386 d-Amphetamine 800 36 5 l-Methamphetamine 8,000 798 10 MDEA 1,000 14414 MDMA 900 526 58 PPA 100,000 59 0.1 Phendimetrazine 100,000 913 0.9

These results show good cross-reactivity not only to d-methamphetaminebut also to ecstasy-class compounds such as MDMA and MBDB.Cross-reactivity with common interfering compounds such as ephedrine,pseudoephedrine, phentermine, and tyramine was minimal, i.e., less than1%.

EXAMPLE 17 Assay Using Conjugate of Present Invention withN-ethylamphetamine Antibody

The conjugate reagent was prepared as described in Example 16.

An antibody-coated microparticle was prepared as described in Example 16using monoclonal antibody NEAMP 48.2 described in U.S. patentapplication Ser. No. 2004/0077021. A microparticle reagent was thenprepared as described in Example 16.

The assay was carried out on a Roche/Hitachi 917 analyzer as describedin Example 16.

Percent cross-reactivity was determined in an immunoassay. A standardcurve was established using known amounts of the target analytemethylenedioxyethylamphetamine (MDEA). Known amounts of variousamphetamine-related drugs were be analyzed as samples in theimmunoassay, and apparent concentrations of these drugs were generatedfrom the standard curve. Apparent concentration of the drug, divided byactual concentration and multiplied by 100, is the percentcross-reactivity of this drug. The standard (dose response) curve thusgenerated is shown in FIG. 12.

The cross-reactivity of the antibody-coated particle with conjugate (12)is given in the table below.

Compound Conc (ng/ml) tested Result % Cross-reactivity d-Amphetamine5000 0 0.0 d-Methamphetamine 1000 89 8.9 l-Methamphetmine 12500 68 0.5MDMA 1000 46 4.6 MDA 5000 37 0.7 MBDB 1000 59 5.9 BDB 5000 47 0.9 PPA200000 70 0.04 l-ephedrine 200000 91 0.05 d-pseudoephedrine 200000 1130.06 Phentermine 200000 103 0.05 Phendimetrazine 100000 44 0.04 Tyramine200000 67 0.03

These results show good cross-reactivity not only tomethylenedioxyethylamphetamine but also cross-reactivity tod-methamphetamine and to ecstasy-class compounds such as MDMA and MBDB.At the same time, cross-reactivity with common interfering compoundssuch as ephedrine, pseudoephedrine, phentermine, and tyramine wasessentially none.

1. A compound having the formula

wherein R₁ is an alkyl linker comprising 2-15 carbon atoms and 0-6heteroatoms, R₂ is a leaving group, and R₃ is a protecting group.
 2. Thecompound of claim 1 wherein R₃ is trifluoroacetyl.
 3. A compound havingthe formula

wherein R₁ is an alkyl linker comprising 2-15 carbon atoms and 0-6heteroatoms, R₂ is aminodextran, and R₃ is a protecting group.
 4. Thecompound of claim 3

wherein R₃ is cleaved and the compound is deprotected.
 5. The compound4-{[4-(3-{(S)-2[methyl-(2,2,2trifluoro-acetyl)-amino]-propyl}-phenoxy)-butyrylamino]-methyl}-benzoicacid2,5-dioxo-pyrrolidin-1-yl ester having the formula


6. The compound having the formula